N-TERMINAL-SPECIFIC ANTI-B-50 (GAP-43) ANTIBODIES INHIBIT CA2-INDUCEDNORADRENALINE RELEASE, B-50 PHOSPHORYLATION AND DEPHOSPHORYLATION, AND CALMODULIN-BINDING()

B-50 (GAP-43) is a presynaptic protein kinase C (PKC) substrate implic ated in the molecular mechanism of noradrenaline release. To evaluate the importance of the PKC phosphorylation site and calmodulin-binding domain of B-50 in the regulation of neurotransmitter release, we intro duced two monoclonal antibodies to B-50 into streptolysin O-permeated synaptosomes isolated from rat cerebral cortex. NM2 antibodies directe d to the N-terminal residues 39-43 of rat B-50 dose-dependently inhibi ted Ca2+-induced radiolabeled and endogenous noradrenaline release fro m permeated synaptosomes. NM6 C-terminal-directed (residues 132-213) a nti-B-50 antibodies were without effect in the same dose range. NM2 in hibited PKC-mediated B-50 phosphorylation at Ser(41) in synaptosomal p lasma membranes and permeated synaptosomes, inhibited P-32-B-50 dephos phorylation by endogenous synaptosomal phosphatases, and inhibited the binding of calmodulin to synaptosomal B-50 in the absence of Ca2+. Si milar concentrations of NM6 did not affect B-50 phosphorylation or dep hosphorylation or B-50/calmodulin binding. We conclude that the N-term inal residues 39-43 of the rat B-50 protein play an important role in the process of Ca2+-induced noradrenaline release, presumably by servi ng as a local calmodulin store that is regulated in a Ca2+ - and phosp horylation-dependent fashion.